Restriction endonucleases and their uses

ABSTRACT

A restriction endonuclease with a recognition sequence 5′-TCGA-3′. The restriction endonuclease is sensitive to the presence of a modified cytosine residue in the recognition sequence. Methods and kits using the restriction endonuclease with a recognition sequence 5′-TCGA-3′ are also disclosed.

This application is a Divisional application of U.S. Ser. No.13/932,365, filed Jul. 1, 2013, which claims priority from co-pendingGreat Britain Application Serial No. GB1212047.3 filed Jul. 5, 2012,each of which is expressly incorporated by reference herein in itsentirety.

FIELD OF INVENTION

The invention relates to cytosine modification sensitive, in oneembodiment cytosine methylation sensitive, restriction endonucleases andtheir uses alone or in combination with other restriction endonucleases,e.g., TaqI, and to methods of analysis of cytosine modification in thecontext of 5′-TCGA-3′ nucleotide sequence.

BACKGROUND

The term epigenetics is used to define heritable changes in generegulation or cellular phenotype that occur without alteration in DNAsequence (Bird 2007). Epigenetic analysis could help explain why cellswith identical genotype could have different phenotype. Thus epigeneticsis considered to be the missing chain between genotype and phenotype(Bemstein et al. 2007; Reik 2007).

The genetic material in eukaryotic cells exists as nucleic acids andprotein complex, termed chromatin. The basic unit of chromatin is thenucleosome, which is composed of DNA wrapped around the octamer ofhistone proteins. Nucleosomes are then packed into higher-orderstructures and finally form chromosomes. Based on the state ofcondensation chromatin is identified as euchromatin (less condensed, andcontaining most actively transcribed genes) or heterochromatin (highlycondensed and the transcriptionally silent form of chromatin). Ingeneral, epigenetic modifications regulate gene expression level throughthe change in chromatin condensation state. DNA methylation usuallysuppresses gene transcription as it induces binding of regulatingproteins, which promote chromatin condensation. Histone modifications(methylation, acetylation, phosphorylation, ubiquitylation, andsumoylation) also regulate chromatin structure and affect DNAtranscription, replication, repair and recombination (Groth et al. 2007;Koch et al. 2007; Krivtsov et al. 2008). For example histone acetylationfavors euchromatin formation as it impairs the DNA-histone interactionand promotes chromatin decondensation. Small and non-coding RNAs canalso regulate chromatin condensation, gene transcription, and thus arealso recognized as epigenetic markers (Mattick and Makunin 2006).

DNA methylation is the most popular and widely analyzed epigeneticmodification, known since 1975 (Holliday and Pugh 1975; Riggs 1975). DNAin mammalian genomes is primarily methylated at CpG sites. Most of CpGsites are located in so called CpG islands. CpG islands are described as0.5 kb-5 kb long, GC rich (>60%) genome regions with a high frequency ofCpG dinucleotides. CpG islands are usually located in the vicinity ofpromoters or the first exons of the genes. Methylation in CpG islandsusually results in transcription inactivation and gene silencing(Robertson and Wolffe 2000).

5-methyl cytosine (m5C) is the most abundant DNA modification in themammalian genome. Often m5C is called the fifth base of DNA. Methylationof cytosine in mammals occurs within CpG sites and is catalyzed by DNAmethyltransferases. De novo methylation of CpG dinucleotides isperformed by DNMT3a and DNMT3b DNA methyltranferases. DNMT1 DNAmethyltransferase maintains the DNA methylation pattern during the DNAreplication cycle (Bird 2002). In addition to 5-methyl cytosine (m5C),one more cytosine modification, 5-hydroxymethyl cytosine (hm5C), wasdiscovered recently in mouse brain cells (Kriaucionis and Heintz 2009).The biological significance of hm5C is still under investigation.However it is hypothesized that hm5C participates in DNA demethylationprocesses and regulation of gene transcription.

The most popular technique used for DNA methylation analysis isbisulfiite treatment. This process deaminates all unmodified cytosinesinto uracils, while m5C and hm5C are resistant to this type ofconversion. During bisulfiite treatment cytosines in the target sequenceare changed to uracil if they are umethylated, or remain unchanged ifthey are methylated. Changes in DNA sequence can further be detectedusing molecular biology techniques such as DNA sequencing, PCR, qPCR,restriction analysis, Southern blotting, primer extension, HPLC, andMALDI-TOF MS etc. (Esteller 2008; Suzuki and Bird 2008).

Along with bisulfite analysis, DNA methylation status can beinterrogated using different restriction endonucleases, whose cleavageof DNA at particular target sequences can be either blocked (Colaneri etal. 2011) or induced (Zheng et al. 2010; Cohen-Kami et al. 2011) by thepresence of methylated cytosine. The level of target DNA digestion isinterrogated using similar techniques as in case of bisulfitemodification. Methylation status comparison between control and testgenomic DNA samples can be performed employing differential methylationhybridization (DMH) technique (Huang et al. 1999). Genomic arrays of CpGislands are used to hybridize genomic DNA digested withmethylation-sensitive endonucleases and amplified by PCR.

A number of restriction endonucleases sensitive to cytosine methylationare available commercially. Many are used in different techniques toassess methylation status of individual CpG targets or methylationsignatures of genomic samples. For example, restriction endonucleasesBstUI (CGCG), HpaII (CCGG), and HhaI (GCGC) were used in differentialmethylation hybridization (DMH) experiments to reveal methylation statusdifferences between control and test samples of genomic DNA (Yan et al.2002). Another example of methylation status analysis also employs theset of four restriction endonucleases sensitive to cytosine methylationAciI (CCGC), HpaII (CCGG), HinP1I (GCGC), and HpyCH4IV (ACGT) and nextgeneration sequencing approach (Colaneri et al. 2011). In both examplesthe authors have used only methylation sensitive restrictionendonuclease digestion analysis.

It is possible to analyze the methylation level of genomic DNA withrestriction enzyme isoschizomer pairs, where the enzymes in the pairhave differing sensitivities to CpG methylation. To date there is onlyone pair of restriction endonucleases, MspI and HpaII, with thesecapabilities. MspI and HpaII are isoschizomers that recognize the targetsequence 5′-CCGG-3′. When the internal CpG in the 5′-CCGG-3′tetranucleotide sequence is methylated cleavage with HpaII is blocked,but cleavage with MspI is not affected. Thus parallel digestions ofgenomic DNA samples with MspI and HpaII can be used to determine themethylation level of the internal cytosine in the CpG base pair locatedin the sequence 5′-CCGG-3′ (Hatada et al. 2006; Khulan et al. 2006; Odaet al. 2009; Takamiya et al. 2009).

This method is only able to examine methylation status in the context of5′-CCGG-3′ and some genes or gene regions of interest, e.g., AT richregions, could have no CCGG sites. Conversely, in CpG rich sequencessuch as gene promoters where regions of CpG islands are located, theremay be many adjacent HpaII/MspI sites, so that methylation analysis ofindividual CpG base pairs employing HpaII/MspI digestion and qPCRquantification could be impaired as even short qPCR amplicons could haveseveral 5′-CCGG-3′ sequences. In particular, after restrictiondigestion, products are analyzed by qPCR with a primer pair flanking theCCGG site of interest. HpaII/MspI sites may be too close to each otherin CpG islands to successfully design amplicon suitable for qPCRanalysis; for optimal PCR efficiency a qPCR amplicon usually is about100 bases length. Moreover, the cleavage of both HpaII and MspI isblocked by m5C modification of the outer cytosine in the 5′-CCGG-3′recognition sequence, and in some cases this could impair theinterpretation of CpG methylation status in the target sequence.

Further analysis methods involve enrichment of DNA carrying methylatedcytosines from the total pool of shared or fragmented DNA usingmethylated DNA immunoprecipitation (MeDIP or mDIP) technique (Weber etal. 2005). Enriched methylated DNA fragments can be further analyzedusing high-throughput DNA analysis methods such as DNA microarrays(MeDIP-chip) or next generation sequencing (MeDIP-seq). Sequencing isthe most informative and preferred analysis technique, whether it isused in combination with bisulfite modification, methylation-sensitiveDNA digestion, or DNA immunoprecipitation. Although next generationsequencing (NGS) prices remain high, continuously increasingcapabilities and decreasing cost of NGS in the near future shouldprovide an opportunity to perform whole genome or at least exomemethylation analysis as a routine approach in diagnostic laboratories.

Despite the above methods, and in view of the awareness of potentialimportance of DNA methylation in phenotype, the need exists for furthertools that can be used to analyze DNA modification status in the rapidlygrowing field of epigenetic research.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a restriction endonucleasewith a recognition sequence 5′-TCGA-3′, which restriction endonucleaseis sensitive to the presence of a modified cytosine residue in therecognition sequence.

The inventors found and characterized a restriction endonuclease thatcan be used to digest DNA comprising the recognition sequence5′-TCGA-3′, and which is sensitive to the presence of a modifiedcytosine residue in the recognition sequence. In particular, therestriction endonuclease is sensitive to the methylation of the cytosineresidue in the recognition sequence, such that when the CpG in the5′-TCGA-3′ is methylated, cleavage with the restriction endonuclease isblocked or significantly reduced. Thus, the restriction endonuclease ofthe invention is useful in the analysis of DNA methylation.

In one embodiment, the invention provides a method for digestingdouble-stranded DNA comprising a recognition sequence 5′-TCGA-3′ inwhich the cytosine residue is unmodified. The method comprises a step ofcontacting the double stranded DNA with the restriction endonuclease.

In one embodiment, the invention provides a method for determining thepresence or absence of a cytosine modification in a recognition sequence5′-TCGA-3′ comprised in double stranded DNA. The method comprises

(a) contacting the double stranded DNA with the restriction endonucleaseto digest the double stranded DNA comprising a recognition sequence inwhich the cytosine residue is unmodified into reaction products; and

(b) determining the presence or absence of reaction products.

The new restriction endonuclease of the invention is an isoschizomer ofthe known enzymes specific for the target recognition sequence5′-TCGA-3′, such as TaqI, but has a different sensitivity to cytosinemodifications, and in particular CpG methylation. As such, the inventiverestriction endonuclease also recognizes the same target sequence5′-TCGA-3′, but when the internal CpG in the 5′-TCGA-3′ tetranucleotidesequence is methylated cleavage with the inventive restrictionendonuclease is blocked or significantly reduced. In contrast cleavagewith the other enzymes and especially TaqI is not significantlyaffected. This presents opportunities for using the inventiverestriction endonuclease in methods of DNA methylation analysis, inparticular in combination with the non-methylation sensitiveisoschizomers.

In one embodiment, the invention provides a method for determining thelevel of methylation in double stranded DNA. The method comprises

(a) contacting a first sample of the double stranded DNA with amethylation sensitive restriction endonuclease as described above todigest the DNA comprising a recognition sequence 5′-TCGA-3′ in which thecytosine residue is unmethylated; and

(b) determining the amount of undigested DNA and/or determining theamount of digested DNA.

In this embodiment the method may also include the additional steps of

(c) contacting a second sample of the double stranded DNA with a secondrestriction endonuclease; and

(d) determining the amount of DNA digestion, where the secondrestriction endonuclease has a recognition sequence 5′-TCGA-3′ and isnot sensitive to methylation of the cytosine residue.

In one embodiment, the invention provides a kit for determining themodification status of a DNA duplex substrate. The kit comprises inseparate containers (a) a restriction endonuclease as described above;and (b) a second restriction endonuclease that has a recognitionsequence 5′-TCGA-3′ and is not sensitive to modification of the cytosineresidue in the recognition sequence. In one embodiment, the secondrestriction endonuclease is TaqI, as indicated above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of a gel showing Lambda DNA (dam⁺, dcm⁺)digestion with TaqI (5′T↓CGA-3′) and HpyF30I.

FIGS. 2A, 2B, 2C show the determination of HpyF30I DNA cleavageposition.

FIG. 3 is a photograph of a gel showing the sensitivity of HpyF30I toDam methylation.

FIG. 4 is a photograph of a gel showing digestion of double-strandedoligonucleotides containing non-modified or m5D modified in both strandsHpyF301 recognition sequence TCGA.

FIGS. 5A, 5B, 5C show amplification of 130 bp DNA fragment performed on5×10⁴ copies of either non-modified (FIG. 5A) or M.SssI methylated (FIG.5B) lambda DNA; with residual amounts (%) of undigested DNA target (FIG.5C).

FIG. 6 is a photograph of a gel showing digestion of PCR fragments withm5C or hm5C modifications in both strands of TCGA sequence.

FIG. 7A, 7B, 7C show spiked PCR fragment digestion analysis in thepresence of human Jurkat cell line genomic DNA.

FIGS. 8A, 8B show methylation analysis of DAPK1 gene promoter region inhuman Jurkat cell line genomic DNA by HpyF301/TaqI/PaeR7I restrictionand subsequent qPCR.

FIGS. 9A, 9B show methylation analysis of RASSFA1 gene promoter regionin human Jurkat cell line genomic DNA by HpyF301/TaqI/PaeR7I restrictionand subsequent qPCR.

FIG. 10 shows digestion of PCR fragments containing C or hm5C in bothDNA strands after treatment with T4 β-glucosyltransferase.

DETAILED DESCRIPTION

FIG. 1 is a gel picture showing Lambda DNA (dam⁺, dcm⁺) digestion withTaqI (5′T↓CGA-3′) and HpyF30I. Lane M: GeneRuler™100 bp Plus DNA Ladder;Lane−: undigested λDNA; Lane H: λ DNA digested with HpyF30I; and Lane T:λ DNA digested with TaqI.

FIGS. 2A to 2C show the determination of HpyF30I DNA cleavage position.55 nt length double-stranded synthetic oligonucleotide (as shown in FIG.2B and FIG. 2C, upper strand SEQ ID NO: 9; lower strand SEQ ID NO: 10)containing one TCGA sequence was digested either with HpyF30I or withTaqI, γ-33P-labeled with T4 DNA polynucleotide Kinase, resolved on 15%PAGE/7% urea gel and visualized using phosphorimager Typhoon Trio. FIG.2A shows the cleavage pattern of digestion of the oligonucleotide withHpyF30I or TaqI (indicated above the gel picture). “−” marks undigestedoligonucleotide. Length of single-stranded oligonucleotides generatedafter cleavage by restriction endonucleases (REs) and separation ondenaturing PAGE are indicated. FIGS. 2B and 2C show the sequence of theoligonucleotide digested with HpyF30I or TaqI, respectively. Therecognition sequence of REs is shown in bold, cleavage positions areshown by arrows. Length of single-stranded oligonucleotides generatedafter digestion with REs and separation on denaturing PAGE areindicated.

FIG. 3 is a gel picture showing the sensitivity of HpyF30I to Dammethylation. pSEAD8 (dam⁺) and pSEAD8 (dam⁻) were digested with HpyF30Ior TaqI and analyzed on 2% agarose gel in TBE buffer. Lane M: GeneRuler™100 bp Plus DNA Ladder Lane−: undigested DNA; Lane H: DNA digested withHpyF30I; Lane T: DNA digested with TaqI. Asterisks denote differences incleavage pattern of dam⁺ and dam⁻ DNA.

FIG. 4 is a gel picture of the digestion of double-strandedoligonucleotides containing non-modified or m5C modified in both strandsHpyF30I recognition sequence TCGA. Double-stranded syntheticoligonucleotide (55 bp) with (a) non-modified or (b) m5C modified inboth strands HpyF30I recognition site was digested with increasingamounts of HpyF30I (indicated on the top of the gel picture). Reactionproducts were analyzed on 10% polyacrylamide gel. Undigestedoligonucleotide (negative control) is indicated by “−” sign. LaneM—GeneRuler™ Ultra Low Range DNA Ladder.

FIGS. 5A to 5C show the amplification of 130 bp DNA fragment performedon 5×10⁴ copies of either non-modified (FIG. 5A) or M.SssI methylated(FIG. 5B) lambda DNA (dam⁻) digested with HpyF30I or TaqI. qPCRreactions were performed on Corbett Rotor-Gene 6000™ (Qiagen)instrument. The amplification plots (FIGS. 5A and 5B) show thedifference in amplification of HpyF30I or TaqI digested or undigestedTCGA target within lambda DNA. NTC denotes amplification curve fromnon-template control. The obtained Cq values were used to calculateresidual amounts (%) of undigested DNA target (FIG. 5C).

FIG. 6 is a gel picture of the digestion of PCR fragments with m5C orhm5C modifications in both strands of TCGA sequence. 1 μg ofdouble-stranded 962 bp PCR fragment with non-modified, m5C methylated orhm5C (hydroxymethylated) recognition site (indicated below the gelpicture) was digested with HpyF30I or TaqI. Reaction products wereanalyzed on 3% agarose gel in TBE buffer. Lane M: GeneRuler™ 50 bp DNALadder; Lane H: DNA digested with HpyF30I; Lane T: DNA digested withTaqI. Undigested DNA (negative control) is indicated by “−” sign.

FIGS. 7A to 7C show spiked PCR fragment digestion analysis in thepresence of human Jurkat cell line genomic DNA. Amplification of 130 bpDNA fragment was performed using 5×10⁴ copies of PCR fragment containingeither C (FIG. 7A) or m5C (FIG. 7B) in both strands digested withHpyF30I or TaqI in the presence of human Jurkat cell line genomic DNA.Reactions were performed on StepOnePlus™ (Applied Biosystems)instrument. The amplification plots and melting curves show thedifference in amplification of digested or undigested TCGA target withinPCR fragment (FIGS. 7A and 7B). NTC denotes amplification curve fromnon-template control. The obtained Cq values were used to calculateresidual amounts (%) of undigested DNA target (FIG. 7C).

FIGS. 8A and 8B show methylation analysis of DAPK1 gene promoter regionin human Jurkat cell line genomic DNA by HpyF30I/TaqI/PaeR7I restrictionand subsequent qPCR. Amplification of 106 bp DNA fragment was performedon 3×10⁴ copies of undigested human Jurkat cell line genomic DNA andgenomic DNA digested with HpyF30I, PaeR7I or TaqI. HpyF30I and TaqIrestriction endonucleases cleave the same TCGA recognition target withinthe analyzed qPCR amplicon. PaeR7I restriction endonuclease cleavesCTCGAG target (with TCGA tetranucleotide inside), is sensitive to innercytosine methylation (REBASE Enzyme No. 1451) and is used as a referenceenzyme to test methylation level of DAPK1 gene. Reactions were performedon StepOnePlus™ (Applied Biosystems) instrument. The amplification plotsshow the difference in amplification of digested or undigested CTCGAGtarget within DAPK1 gene promoter region (FIG. 8A). NTC denotesamplification curve from non-template control. The obtained Cq valueswere used to calculate residual amounts (%) of undigested DNA target(FIG. 8B).

FIGS. 9A and 9B show methylation analysis of RASSFA1 gene promoterregion in human Jurkat cell line genomic DNA by HpyF30I/TaqI/PaeR7Irestriction and subsequent qPCR. Amplification of 150 bp DNA fragmentwas performed on 3×10⁴ copies of undigested human Jurkat cell linegenomic DNA and genomic DNA digested with HpyF30I, PaeR7I or TaqI.HpyF30I and TaqI restriction endonucleases cleave the same TCGArecognition target within the analyzed qPCR amplicon. PaeR7I restrictionendonuclease cleaves CTCGAG target (with TCGA tetranucleotide inside),is sensitive to inner cytosine methylation (REBASE) and is used as areference enzyme to test methylation level of RASSFA1 gene promoterregion. Reactions were performed on StepOnePlus™ (Applied Biosystems)instrument. The amplification plots show the difference in amplificationof digested or undigested CTCGAG target within RASSFA1 gene promoterregion (FIG. 9A). NTC denotes amplification curve from non-templatecontrol. The obtained Cq values were used to calculate residual amounts(%) of undigested DNA target (FIG. 9B).

FIG. 10 shows digestion of PCR fragments containing C or hm5C in bothDNA strands after treatment with T4 β-glucosyltransferase (T4 BGT).Double-stranded 1095 bp length PCR fragments with non-modified C or hm5C(hydroxymethylated) in both strands (indicated below the gel picture)were treated with T4 BGT (control reactions were incubated without T4BGT) and subsequently digested with TaqI, HpyF30I or FastDigest™Mfel.Reaction products were analyzed on 2% agarose gel in TBE buffer. M:GeneRuler™ 50 bp DNA Ladder; T: DNA digested with TaqI; H: DNA digestedwith HpyF30I; F: DNA digested with FastDigest™Mfel. Undigested DNA(negative control) is indicated by “−” sign.

The invention provides a restriction endonuclease (restriction enzyme)with a recognition sequence or target sequence 5′-TCGA-3′, whichrestriction endonuclease is sensitive to the presence of a modifiedcytosine residue in the recognition sequence.

The modified cytosine residue may be a natural modification, such asN4-methylcytosine, 5-methylcytosine, 5-hydroxymethylcytosine,glucosylated-hydroxymethylcytosine, or 5-carboxylcytosine (5caC), or maybe a synthetic modification such as 3-methylcytosine, 5-ethynylcytosineor 5-phenylcytosine. In one embodiment, the modified cytosine residue isnot N4-methylcytosine.

In one embodiment, the cytosine is modified at position 5 or themodification comprises a methyl group. In one embodiment, the modifiedcytosine residue is a methylated cytosine residue and the cytosine ismethylated at position 5, i.e. the cytosine is modified with a groupthat includes a methyl group at position 5. In one embodiment, themethylated cytosine is 5-methylcytosine (m5C), 5-hydroxymethylcytosine(hm5C), or glycosylated 5-hydroxymethylcytosine.

The restriction endonuclease is modification sensitive such that whenthe cytosine in the recognition sequence is modified the endonucleasehas a reduced ability to cleave the sequence, i.e. the endonuclease'scleavage of the recognition sequence 5′-TCGA-3′ is blocked or impairedby the cytosine modification. In one embodiment, an impaired ability tocleave the recognition sequence means that under conditions at which therestriction endonuclease is most active, optimal reaction conditions,the presence of the modified cytosine residue in the recognitionsequence reduces the endonuclease's ability to cleave the sequence bymore than 80% in one embodiment, and more than 90% in one embodiment. Inone embodiment when there is a modified cytosine residue in therecognition sequence, e.g., a methylated cytosine residue such as 5mC orhm5C, the endonuclease cleaves ≦5% of the recognition sequences present.The percent of DNA cleavage can be determined by qPCR analysis.

Restriction endonucleases according to the invention can be obtainedfrom Helicobacter species, in one embodiment Helicobacter pylori.Suitable species and strains are publicly available, and presence of theinventive enzyme in the strain can be determined by testing theenzymatic activity of crude cell extracts.

A restriction endonuclease according to the invention may comprise anamino acid sequence SEQ ID NO: 4, or a sequence having at least 70%identity, at least 80% identity, at least 85% identity, at least 90%identity, or at least 95% identity therewith.

SEQ ID NO: 4 MQFLNQSLGFFNKGHFEPIDRNFITESYQALKPIEEIQNKYNKHDNDSFLNELRDSMVALYLDYELINIQKHGLDAKRSSSDEFLEIKQVSFQSKTWSATFNDTTLEKAKVFCDIKTTLAVGVWNNISNLLFIVYGKHPEIGLYLEQKVKECHNESRRSTQTIGVSKLIKEFDFKMKPIDLKEQELINLFNLKFGHFSWE NHLA

A restriction endonuclease according to the invention may be encoded bythe nucleotide sequence SEQ ID NO: 3 (the nucleotide sequence of thehpyF30IR gene) or a nucleotide sequence having at least 70% identity, atleast 80% identity, at least 85% identity, at least 90% identity, or atleast 95% identity therewith.

SEQ ID NO: 3 atgcaatttttaaatcaatctttgggattttttaataaagggcactttgagcccattgacagaaacttcatcacagaaagctatcaagcactaaagccgattgaagaaattcaaaataaatacaataaacatgacaacgattcatttttgaatgaattgagagatagcatggtggctctatatttagattatgagcttatcaatattcaaaagcatggtcttgatgccaaaagaagttcaagcgatgaatttttagaaatcaaacaagtgtcctttcaaagtaaaacttggagcgcgacttttaatgacaccactttagaaaaagccaaagttttttgcgatattaaaacgactttagccgtgggcgtttggaataatatttctaatcttttattcattgtttatggaaagcaccctgaaattggcttgtatttagaacaaaaagtaaaagagtgtcataatgagagcaggcgttcaactcaaacgataggggttagtaaattgatcaaagaatttgattttaaaatgaaacccattgatttaaaagaacaagagcttatcaatctttttaatcttaaatttggtcatttttcttgggaa aaccatcttgcataa

Sequence variants are described below, but in general variations to theamino acid and nucleotide sequence can be made that result inconservative amino acid substitutions. Structurally and functionallysignificant areas of the enzyme can be identified by mutational and/orstructural analysis methods known in the art, or can be predicted fromamino acids sequence alignment with sequences of other functionallysimilar enzymes (Orlowski & Bujnicki, 2008).

In one embodiment the restriction endonuclease is HpyF30I, having theamino acid sequence SEQ ID NO: 4. However, the sequence of otherrestriction endonucleases according to the invention are provided inGenbank, e.g.,

ADO05010.1 - hypothetical protein HPSAT_01305 [Helicobacter pylori Sat464](SEQ ID No: 11): 1mqflnqslgf fnkghfepid rnfitesyqa lkpieeiqnk ynkhdndsfl nelrdsmval 61yldyeliniq khgldakrss sdefleikqv sfqsktwsat fndttlekak vfcdikttla 121vgvwnnisnl lfivygkhpk igly1eqkvk echnesrrst qtigvsklik kfdfkmkpid 181lkeqelinlf nlkfghfswe nhlaAEN17973.1 - hypothetical protein HPPN135_01350 [Helicobacter pylori Puno135](SEQ ID No: 12): 1mqflnqslqf fnkghfkpid rnfitesyqa lkpieeiqnk ynkhdndsfl nelrdsmval 61yldyeliniq khgldakrss sdefleikqv sfqsktwsat fndttlekak vfcdikttla 121vgvwnnisnl lfivygkhpk ig1y1eqkak echnesrrst qtigvsklik efdfkmkpid 181lkeqelinlf nlkfghfswe nhlaACX97473.1 - hypothetical protein KHP_0259 [Helicobacter pylori 51](and BAJ54865.1 - hypothetical protein HPF16_0268 [Helicobacter pylori F16])(SEQ ID No: 13): 1mqflnqslgf fnkghfepid rnfiaesyqa lkpieeiqnk ynkhdndsfl nelrdsmval 61yldyeliniq khgldakrsl sdefleikqv sfqsktwsat fndttlekak vfcdikttla 121vgvwnnisnl lfivygkhpk iglyleqkvk echnesrrst qtigvsklik efdfkmkpid 181lkeqelinlf nlkfghfswe nhlaADO03503.1 - hypothetical protein HPCU_01630 [Helicobacter pylori Cuz20](SEQ ID No: 14): 1mqflnqslgf fnkghfepid rnfitesyqa lksieeiqnk ynkhdndsfl nelrdsmval 61yldyeliniq khgldakrns sdefleikqv sfqsktwsat fndttlekak vfcdikttla 121vgvwnnisnl lfivygkhpk iglyleqkvk echnesrrst qtigvsklik efdfkmkpid 181lkeqelinlf nlkfghfswe nhlaBAJ57132.1 - hypothetical protein HPF30_1035 [Helicobacter pylori F30](SEQ ID No: 15): 1mqflnqslgf fnkghfepid rtfitesyqa lkpiekiqnk ynkhdndsfl nelrdsmval 61yldyeliniq khgldatrss sdefleikqv sfqsktwsat fndttlekak vfcdikttla 121vgvwnnisnl lfivygkhpk iglyleqkvk echnesrrst qtigvsklik efdfkmkpid 181lkeqelinlf nlkfghfswe nhlaBAJ57852.1 - hypothetical protein HPF32_0270 [Helicobacter pylori F32](SEQ ID No: 16): 1mrflnqslgf fnkgcfepid rnfiaesyqa lkpieeiqnk ynkhdndsfl nelrdsmval 61yldyeliniq khgldakrss sdefleikqv sfqsktwsat fndttlekak vfcdikttla 121vgvwnnisnl lfivygkhpk iglyleqkvk echnesrrst qtigisklik efdfkmkpid 181lkeqelinlf nlkfghfswe nhlaACX98882.1 - hypothetical protein HPKB_0272 [Helicobacter pylori 52](SEQ ID No: 17): 1mrflnqslgf fnkgrfepid rnfitesyqa lkpieeiqnk ynkhdndsfl nelrdsmval 61yldyeliniq khgldakrss sdefletkqv sfqsktwsat fndttlekak vfcdvkttla 121vgvwnnisnl lfivygkhpe ivlyleqkvk echnesrrst qtigvsklik efdfkmkpid 181lkeqelinlf nlkfghfswe nhlaZP_03438878.1 - hypothetical protein HP9810_1g62 [Helicobacter pylori 98-10](and EEC23581.1 - hypothetical protein HP9810_1g62 [Helicobacter pylori 98-10])(SEQ ID No: 18): 1mqflnqslgf fnkghfepid rnfiaesyqa lkpieeiqnk ynkhdndsfl nelrnsival 61yldyeliniq khgldakrss sdefleikqv sfqsktwsat fndttlekak vfcdikttla 121vgvwnnisnl lfivygkhpk iglyleqkvk echnesrhst qtigvskltk efdfkmkpid 181lkeqelinlf nlkfghfswe nhlaZP 03436984.1 - hypothetical protein HPB128 21g37 [Helicobacter pylori B128](and YP_003729323.1 - hypothetical protein HPB8 71302 [Helicobacter pylori B8],EEC25275.1 - hypothetical protein HPB128_21g37 [Helicobacter pylori B128]andCBI66859.1 - conserved hypothetical protein [Helicobacter pylori B8])(SEQ ID No: 19):1 mqflnqslgf fnkgcfepid rnfitesyqa lkpieeiqnk ynkhdndsfl nelrdsmval 61yldyeliniq khgldakrss sdefleikqv sfqsktwsat fndttlekak vfcdikttla 121vgvwnnisnl lfivygkhpe iglyleqkvk echnesrrst qtigvsklik efdfkmkpid 181lkeqelinlf nlkfghfsADU84288.1 - hypothetical protein HPSA_01320 [Helicobacter pylori SouthAfrica7](SEQ ID No: 20): 1mqflnqslgf fnkgcfepid rnfitesyqa lkpiekiqnk ynkhdndsfl nelrdsmval 61yldyelintq khgldakrss sdefleikqv ffqsktwsat fndttlekak vfcdikttla 121vgvwnnisnl lfivygkhpe mg1yleqkvk echnesrrst qtigisklik efdfkmkpid 181skeqelinlf nlkfgrfswe nylaADU81143.1 - hypothetical protein HPGAM_01475 [Helicobacter pylori Gambia94/24](SEQ ID No: 21): 1mqflngslgf fnkgcfepid rnfitesyqa lkpieeiqnk ynkhdndsfl nelrdsmval 61y1dynlintq khgldakrss sdefleikqv sfqsktwsat fndttlekak vfcdikttla 121vgvwnnisnl lfivygkhpe mg1yleqkvk echnesrrst qtigisklik efefkmkpid 181skeqelinlf nlkfgrfswe nclaYP_002265873.1 -hypothetical protein HPG27_240 [Helicobacter pylori G27](AC127007.1 - hypothetical protein HPG27_240 [Helicobacter pylori G27]andGENE ID: 6963126 HPG27_240 - hypothetical protein [Helicobacter pylori G27])(SEQ ID No: 22): 1mqflnqs1gf fnkgcfepid rnfitesyqa lkpieeignk ynkhdndsfl nelrdsmval 61yldyd1intq khgldakrss ndefleikqv sfqsktwsat fndttlekak vfcdikttla 121vgvwnnisnl lfivygkhpe mglyleqkvk echnerrrst qtigisklik efdskmkpid 181skeqelin1f nlkfgrfswe nylaYP_665084.1 - hypothetical protein Hac_1347 [Helicobacter acinonychis str. Sheeba](and CAK00085.1 - hypothetical protein Hac_1347 [Helicobacter acinonychis str.Sheeba)(SEQ ID No: 23): 1mqflnqs1df fnkgcfepid rnfitescqa lkpiekiqnk ynkhdndsff nelrdsmval 61fldydlintq khgldakkvq gnefleikqv sfqsktwsat fndttlekak vfcdikttla 121vgvwnnisnl lfivygkhpe iglyleqkvk echngsrrst qtigisklik efsfkmkpid 181lkeqelin1f klkfgnfswg nylaEGQ80805.1 - hypothetical protein HMPREF9094_0161 [Fusobacterium nucleatum subsp.animalis ATCC 51191](SEQ ID No: 24): 1mwenreekkm fknqnlplfn kgtykeidrv yvsrvqnamf qvmelqeeyg kydndsflne 61mkdsmvgmyl gyefvnidkh gfdakrksnk ydewlevkqv sfkseswqat fndttiekae 121afkdiklnla vgvwnkmmel mfivygknye igeylekmvi kckeeqrrst qtisvqslie 181nynfrvkpvn nsskeveell kirfkkynwr dridkeZP_06753958.1 - conserved hypothetical protein [Simonsiella muelleri ATCC 29453](and EFG30885.1 - conserved hypothetical protein [Simonsiella muelleri ATCC 29453)(SEQ ID No: 25): 1mteqvffknq dlkifhkgny qeinrdfise anhaikplnv iqkkykkldn dtffnelrdg 61migaylgydl vniekhglda knsennqfle vkqasfsaks wvatfndtty ekaeafedek 121lflavgvwag lsellfivyg qnpliggylk srvdifksgg svrstqsiti kdlvvcygfk 181ilfptqerke iknifrlkyk gedwwsgafv de

Other embodiments of the restriction endonuclease of the invention arethat it is a neoschizomer of Taq I, and in one embodiment cuts in themiddle of the recognition/target sequence to form blunt ended fragments,(i.e. 5′-TC↓GA-3′. In addition, or alternatively, the cleavage of therestriction endonuclease is blocked by methylation of adenine (m6A) inthe recognition sequence.

In one embodiment a composition comprises the restriction endonucleasedescribed above and a buffer. The buffer is suitable for long termstable storage of the restriction endonuclease. Suitable components forendonuclease storage buffers are known in the art. The storage buffermay comprise potassium phosphate, NaCl, EDTA, DTT, Triton X-100, BSAand/or glycerol.

The invention also provides an isolated polynucleotide encoding therestriction endonuclease of the invention. The polynucleotide may be DNAor RNA. The polynucleotide may comprise SEQ ID NO: 3, or a sequencehaving at least 70% identity, at least 80%, at least 85%, at least 90%identity, or at least 95% identity therewith.

In one embodiment, the isolated polynucleotide of the invention is in avector, a carrier vehicle that can be used to transfer thepolynucleotide into a host cell. In one embodiment, the polynucleotidein the vector is under the control of an inducible promoter. In oneembodiment, the invention provides a host cell that is resistant tocleavage by the restriction endonuclease of the invention, with thevector. The host cell can be used in the production of the restrictionendonuclease. A suitable production method comprises culturing the hostcell under conditions that allow for the expression of the restrictionendonuclease. Suitable vectors, host cells, methods of production, andconditions for expression are known in the art. The vector can be aplasmid, such as pET. Suitable host cells include E. coli. In oneembodiment, the host cell should express a suitable methyltransferase toprotect the host genome against cleavage by the restrictionendonuclease. A suitable methyltransferase is M.HpyF30I having SEQ IDNo: 8 or encoded by the nucleotide sequence of the hpyF30IM gene havingSEQ ID No:7.

SEO ID NO: 8 MHKVFIMEALECLKRIEKESIQTIYIDPPYNTKSSNFEYEDAHADYEKWIEEHLILAKSVLKQSGCIFISIDDNKMAEVKIIANEIFGTRNFLGTFITKQATRSNAKHINITHEYVLSYAKNKAFAPGFKILRTLLPIYAKALKDLMRTIKNVFRQKGQAQAQLVLKEQIKELSKKEHFNFLKNYNLVDEKGEIYFAKDLSTPSHPRSVAIOEINLFLEPLKSRGWSSDEKLKDLYYQNRLIFKNNRPYEKYYLKESQDNCLSVLDFYSROGTKDLEKLGLKGLEKTPKPVGLIKYLLLCSTPKDSIILDFFAGSGTTAQAVIEANRDYDLNWSFYLCQKEEKIKNNPQAVSILKNKGYQNTISNIMLLRLEKIIKRSEYEILR SEQ ID NO: 7atgcataaagtttttatcatggaagctttggaatgtttgaaaagaatagaaaaagaaagcatccaaaccatctatatagaccccccttataacactaagagttctaactttgaatatgaagacgctcatgctgactatgaaaaatggattgaagaacacttgattttagcaaagtctgtgttaaaacaaagcggttgtatttttatttctatagatgacaataaaatggctgaagttaaaatcattgccaatgaaatttttggaacgcgcaattttttaggcacttttatcactaaacaagccacaaggtctaacgctaaacacatcaatattacccatgaatatgttttaagctacgccaaaaataaagcgttcgctcctggttttaaaatcttacgaacgcttttgcccatttatgctaaagcattaaaagatttaatgcgaacgattaaaaatgtttttagacaaaaaggacaagctcaagcccaacttgtcctaaaagaacaaatcaaagagttatctaaaaaagaacattttaattttttaaaaaattataatttggtggatgaaaaaggtgaaatttatttcgctaaagatttatctacgccttcacacccacgcagtgtagcgatacaagaaatcaatctttttttagaacccttaaaaagcagagggtggagcagcgatgaaaagcttaaggatttatattatcaaaacagacttatttttaagaacaatcgcccttatgaaaaatattacctaaaagaatcgcaagataattgtttgagcgtgttggatttttatagccgacaaggcacaaaagatttagaaaaattaggcctaaaggggctttttaagacgccaaaacctgtaggattgattaaatatttattgttatgctccacccctaaagattctattattttagatttttttgcaggcagtgggacaacagcgcaagcggttatagaagctaatagggattatgatttgaattggtctttttatttgtgtcaaaaagaagaaaaaattaaaaataacccgcaagctgttagcattttaaaaaacaaggggtatcaaaacacgatttcaaacatcatgctgttgcgtttagaaaagatcatcaaaagaagtgaatacgaaattttaa gataa

The restriction endonuclease of the invention can be used to cleave DNAcomprising the recognition sequence. In one embodiment, the inventionprovides the use of a restriction endonuclease described above to digestdouble-stranded DNA, where the double-stranded DNA comprises arecognition sequence 5′-TCGA-3′ in which the cytosine residue isunmodified. Alternatively in one embodiment the invention is a methodfor digesting double-stranded DNA comprising a recognition sequence5′-TCGA-3′ in which the cytosine residue is unmodified; the methodcomprises a step of contacting the double stranded DNA with arestriction endonuclease as described above.

The restriction endonuclease can be used to determine the presence orabsence of a cytosine modification as described above in a recognitionsequence 5′-TCGA-3′ comprised in double stranded DNA. The doublestranded DNA is contacted with the restriction endonuclease to digestthe double stranded DNA comprising a recognition sequence in which thecytosine residue is unmodified into reaction products, and the presenceor absence of reaction products is assessed to determine the presence orabsence of the cytosine methylation. The presence of reaction productsmay be detected, e.g., using gel electrophoresis.

In one embodiment, the restriction endonuclease can be used to determinethe level of cytosine modification, e.g., the level of cytosinemethylation, in double stranded DNA. A first sample of the doublestranded DNA is treated with the restriction endonuclease to digest theDNA comprising a recognition sequence 5′-TCGA-3′ in which the cytosineresidue is unmodified, and the level of modification is determined basedon the amount of digested DNA and/or the amount of undigested DNA. Theamount of digested and/or undigested DNA can be determined using methodsknown in the art, such as qPCR.

The sample of double stranded DNA is not limited, but in one embodimentit is a sample of genomic DNA.

In one embodiment, the restriction endonuclease can be used incombination with restriction endonuclease (a second restrictionendonuclease) that is not cytosine modification sensitive, especiallyone that is not sensitive to the presence of cytosine methylation, e.g.5-methylcytosine (m5C) or 5-hydroxymethylcytosine (hm5C)) in therecognition sequence. A further digestion with a second sample of thedouble stranded DNA is performed with the second restrictionendonuclease. The second restriction endonuclease is an isoschizomer ofthe restriction endonuclease of the invention. The second restrictionendonuclease can be any enzyme that has the recognition sequence5′-TCGA-3′ but that is not sensitive to cytosine modification, e.g. notCpG methylation sensitive. In one embodiment, the second restrictionendonuclease is TaqI or a mutant thereof. The restriction endonucleaseTaq I and mutants thereof are known in the art. The amino acid and genesequence of TaqI restriction endonuclease are publicly available, e.g.amino acid sequence EMBL No.: AAA27505. The second restrictionendonuclease can be one with an amino acid sequence that has at least80%, at least 85%, or at least 90% sequence identity with SEQ ID No: 26.

SEQ ID No: 26: 1mastqaqkal etferflasl dlesyqqkyr piktveqdlp relnplpdly ehywkaledn 61psflgfeeff dhwwekrlrp ldefirkyfw gcsyafvrlg learlyrtav siwtqfhfcy 121rwnascelpl eaapeldaqg idalihtsgs stgiqikket yrseaksenr flrkqrgtal 181ieipytlqtp eeleekakra rvngetyrlw akvahhldrl engfvifres yvksielflq 241knaptlsgli rwdrvaqeal tap

The second restriction endonuclease may be TfiTok6A1I, TflI, Tsp32I, orTthB8I (REBASE), which share a high sequence homology with TaqI.

After cleavage of the first and second samples with either therestriction endonuclease of the invention or second restrictionendonucleases, i.e., the isoschizomer, the residual amount of undigestedDNA can be evaluated using, e.g., qPCR analysis. The total amount oftarget DNA in the first and second samples can be calculated from theundigested DNA sample. The amount of methylated target is calculatedfrom the first sample digested with the enzyme of the invention. Thesecond sample digested with the second restriction endonuclease (e.g.TaqI) is used as a positive control for DNA digestion completeness.

Such a method provides the possibility to identify the methylation level(0%-100%) of cytosine in the CpG base pair located in the middle of aTCGA recognition sequence. For this type of analysis to be accuratelyperformed, two restriction endonucleases recognizing the same sequenceand differing in sensitivity to CpG methylation are required.Previously, as mentioned above, there has only been one pair ofrestriction endonucleases used in combination for methylation statusanalysis, i.e. MspI and HpaII, which have the recognition site5′-CCGG-3′. The methods of the invention involving the combination ofenzymes described above is an alternative to HpaII and MspI (recognitionsequence 5′-CCGG-3′) pair of restriction endonucleases and allows theinvestigation of methylation state of CpG dinucleotides present in thegenome in a different sequence context, thus greatly expanding theassortment of available molecular tools for epigenetic analysis. Similarmethods to those currently carried out with HpaII and MspI (Hatada etal. 2006; Khulan et al. 2006; Oda et al. 2009; Takamiya et al. 2009) canalso be carried out with the combination of the restriction endonucleaseof the invention and the CpG methylation insensitive isoschizomers.Thus, alternative genes and gene regions, e.g., AT rich regions thatpotentially have no CCGG sites, or gene promoters that are typically CpGrich sequences where there may be too many CCGG sites, are available formethylation analysis. The difference between methylation of 5′-CCGG-3′sites and 5′-TCGA-3′ sites within the same genes/gene regions can beinvestigated using the inventive methods in combination with parallelmethods involving HpaII and MspI, or simultaneous restriction analysisusing HpaII/MspI, and the inventive enzyme pair could be used toincrease qPCR assay sensitivity. In this case several restrictiontargets, CCGG and/or TCGA, located in close proximity may beinterrogated in the analyzed amplicon of qPCR. If the DNA region ofinterest contains CCGG and TCGA sequences in close proximity, it ispossible to digest DNA with HpaII/MspI and the enzyme pair of theinvention, and subsequently analyze reaction products in qPCR using thesame pair of primers flanking these targets, and to determine CpGmethylation level of CpGs located close to each other.

In one embodiment, the invention provides a kit for determining themodification status of a DNA duplex substrate. The kit comprises inseparate containers (a) a restriction endonuclease according to theinvention; and (b) a second restriction endonuclease. The restrictionendonuclease and the second restriction endonuclease of the kit are asdescribed above. In one embodiment, the restriction endonuclease of theinvention is sensitive to methylation of the cytosine residue in therecognition sequence 5′-TCGA-3′, while the second restrictionendonuclease is an isoschizomer and is not sensitive to methylation ofthe cytosine residue. In one embodiment, the second restrictionendonuclease is TaqI.

In one embodiment, the kit further comprises a container of genomic DNA.This can be used for a control reaction involving the enzymes in thekit.

In one embodiment, the kit further comprises one or more containers ofreaction buffer for the restriction endonuclease of the invention and/orthe second restriction endonuclease. Suitable reaction buffers are knownin the art and may comprise Tris-HCl, Tris-acetate, MgCl₂, Mg-acetate,K-acetate, NaCl, and/or BSA.

The invention will now be described in further detail, by way of exampleonly, with reference to the following Examples and related Figures.

Example 1 Restriction Endonuclease HpyF30I

Cloning of Restriction Endonuclease HpyF30I from Helicobacter pyloriRFL30 Strain

The gene of HpyF30I restriction endonuclease (REBASE Enz Num 4343) wasPCR amplified from H. pylori strain RFL30 genomic DNA template usingpair of primers, Tq-hpR-dir (GCTATTTAAATGCAATTTTTAAATCAATCTTTGGGAT (SEQID No: 1)) and Tq-hpR-rev (ATAGCGGCCGCTTATGCAAGATGGTTTTCCCAAG (SEQ IDNo: 2)), designed according to the sequence information of Helicobacterpylori strain 26695, i.e. the primers were complementary to the 5′ and3′ ends of HP0261 ORF. The HP0261 ORF is adjacent to the gene HP0260.Gene HP0260 (hpyF30IM) has previously been found to encode aTCGA-specific methyltransferase M.HpyAORF260 (Vitkute et al. 2001). Apair of primers designed to be complementary to the 5′ and 3′ ends ofgene encoding DNA methyltransferase M.HpyAORF260 from Helicobacterpylori 26695 strain, and which were used to amplify the hpyF30IM genefrom H. pylori RFL30 strain are Tq-hpM-dir1(CTGCAGAAGGAGATTTAAATGCATAAAGTTTTTATCATGGAAG (SEQ ID No: 5)) andTq-hpM-rev (ATAGCGGCCGCTTATCTTAAAATTTCGTATTCACTTCT (SEQ ID No: 6)). Thenucleotide sequence of hpyF30IM (SEQ ID No: 7) and amino acid sequence(SEQ ID No: 8) of M.HpyF30I are provided above. The amino acid sequenceof M.HpyF30I is almost identical to M.HpyAORF260, i.e., has 96% ofidentical amino acids, which is proved to recognize the same sequence asM. TaqI methyltransferase. According to the sequence analysismethyltransferase M.HpyF30I is proposed to be m6A methyltransferase(Vitkute et al. 2001).

The restriction endonuclease gene was cloned into pET type expressionvector and was expressed in E. coli ER2566 strain in the background ofmethyltransferase M.HpyF30I (pACYC184). Nucleotide sequence of hpyF30IR(SEQ ID No: 3) and amino acid sequence of HpyF30I restrictionendonuclease (SEQ ID No: 4) are provided above.

Restriction endonuclease HpyF30I was purified to about 90% purityaccording to SDS-PAGE using several subsequent ion-exchange and affinitychromatography steps. Absence of endo and exonucleases was confirmedusing standard labeled oligonucleotide (LO) quality test(http://www.fermentas.com/en/support/technical-reference/restriction-enzymes/quality).

Identification of Other TCGA Specific Methylation Sensitive Enzymes

BLAST search has identified many highly homologous proteins (up to 99%of identical amino acids) in different strains of Helicobacter pylori,which are identified as hypothetical proteins in Genbank.

The Specificity of HpyF30I Restriction Endonuclease

E. coli genomic DNA in vivo methylated with M.HpyF30I (putativespecificity TCGA) was protected against the cleavage of HpyF30Irestriction endonuclease. The lambda DNA cleavage pattern with TaqI(5′-T↓CGA-3′ prototype restriction endonuclease) and HpyF30I restrictionendonucleases are identical (FIG. 1). These data indirectly confirm thatHpyF30I restriction endonuclease recognizes the same 5′-TCGA-3′ sequenceas the prototype restriction endonuclease TaqI. Recognition sequence ofHpyF30I restriction endonuclease was directly confirmed indouble-stranded synthetic oligonucleotide digestion experiments used toidentify the cleavage position of HpyF30I restriction endonuclease(FIGS. 2A to 2C). Even though HpyF30I restriction endonucleaserecognition sequence is the same as TaqI, the cleavage position isdifferent. Prototype restriction endonuclease TaqI cleaves TCGA sequenceafter the T base and produces two nucleotide long cohesive ends (FIG.2C). Restriction endonuclease HpyF30I cleaves TCGA sequence after the Cbase (in the middle of recognition sequence) and produces the DNA withblunt ends (FIG. 2B).

Methods and Materials

Lambda DNA digestion. 1 μg of λ DNA (dam⁺, dcm⁺) was digested inparallel with 10 units of HpyF30I REase in 30 μl reaction buffer Tango™(33 mM Tris-acetate; 10 mM Mg acetate; 66 mM K-acetate; 0.1 mg/ml BSA;pH 7.9 at 37° C.) at 37° C. for one h and with TaqI REase in reactionbuffer TaqI (10 mM Tris-HCl; 5 mM MgCl₂; 100 mM NaCl; 0.1 mg/ml BSA; pH8.0 at 37° C.) at 65° C. for one h. The reactions were stopped by adding6×DNA Loading Dye & SDS Solution and resolved on 1.7% agarose gel in TBEbuffer.

Identification of DNA cleavage position by HpyF30I restrictionendonuclease. To determine DNA cleavage position of HpyF30I, 1 μg 55 ntlength double-stranded synthetic oligonucleotide (obtained afterannealing of two complementary single-stranded oligonucleotides)containing one TCGA sequence was digested in parallel with 10 units ofHpyF30I REase in 25 μl reaction buffer Tango™ at 37° C. for 1 h and withTaqI REase in TaqI reaction buffer at 65° C. for 1 h. Ten μl of reactionproducts were γ-³³P-labeled with T4 DNA polynucleotide kinase (usingprotocol recommended by manufacturer) in 20 μl total volume of reactionmixture containing 50 mM imidazole-HCl (pH 6.4 at 25° C.), 18 mM MgCl₂,50 mM DTT, 0.1 mM spermidine, 0.1 mM ADP, 10 pmol γ-ATP, 10 pmol PEG6000, and 1 u of the enzyme. Reactions were performed at 37° C. for 30min and stopped by heating at 75° C. for 10 min. Undigestedoligonucleotide was γ-³³P-labeled using the same reaction conditions andused as negative control. After addition of 2×RNA Loading Dye sampleswere heated at 95° C. for five min. Ten μl of each were separated on 15%PAGE/7% urea gel in TBE buffer and visualized using phosphorimagerTyphoon Trio.

Example 2 Methylation Sensitivity of HpyF30I Restriction Endonuclease

The M.HpyF30I is putative m6A methyltransferase of TCGA specificity.Because E. coli DNA in vivo methylated with M.HpyF30I is completelyprotected against cleavage of HpyF30I, it was deduced that restrictionendonuclease is sensitive to m6A modification in TCGA recognitionsequence. Restriction endonuclease HpyF30I sensitivity to m6Amodification was directly confirmed in DNA digestion experiment withoverlapping Dam methylation (FIG. 3). Dam methylation target GATC mayoverlap the HpyF30I recognition target TCGA in the context of TCGATCsequence. pSEAD8 plasmid DNA, which contains 11 TCGA sequences, one ofthem overlapping with dam sequence in one strand and the other one inboth DNA strands was purified from E. coli dam⁺ strain JM109 anddigested with TaqI restriction endonuclease (sensitive to m6Amodification) and HpyF30I. DNA cleavage pattern was identical for bothenzymes and differed from cleavage pattern of pSEAD8 isolated from dam⁻E. coli strain GM2163 (FIG. 3). The result indicated that HpyF30Irestriction endonuclease likewise TaqI did not cleave hemimethylated andfully methylated TCGm6A targets.

Sensitivity of HpyF30I restriction endonuclease to m5C and hm5C wastested in double-stranded synthetic oligonucleotide digestion, M.SssImethylated lambda DNA digestion and PCR fragment DNA (synthesized withdCTP or dm5CTP, or dhm5CTP) digestion experiments. Double-strandedsynthetic oligonucleotide having TCGA recognition site with unmodified Cwas digested with HpyF30I restriction endonuclease while theoligonucleotide having TCGA sequence with m5C in both strands wascompletely resistant to HpyF30I treatment even with the highest enzymeamounts used in the experiment (FIG. 4).

Lambda DNA (dam⁻) and lambda DNA (dam⁻) methylated with M.SssImethyltransferase were used to interrogate HpyF30I ability to cleavecompletely modified TCGA target (m5C present in both DNA strands).Cleavage completeness was evaluated using qPCR performed on dedicatedTCGA target within amplification region (FIGS. 5A to 5C). Methylatedlambda DNA was completely resistant to HpyF30I cleavage (100% ofresidual DNA target amplification after restriction reaction), meanwhileonly minor amount of undigested target was detected (0.2%) after thetreatment with TaqI restriction endonuclease. In the same experimentunmodified lambda DNA target was efficiently digested with both HpyF30I(0.6% of residual DNA target left) and TaqI (0.06%) enzymes.

PCR fragments synthesized using dCTP, dm5CTP or dhm5CTP were used tocheck HpyF30I sensitivity to C base modifications inside the TCGArecognition sequence (FIG. 6). Ordinary PCR fragment (synthesized withdCTP) was completely digested with HpyF30I restriction endonuclease.Other two PCR fragments (synthesized with dm5CTP or dhm5CTP) could bedigested only with TaqI restriction endonuclease, which is not sensitiveto C base modification within TCGA sequence, but not with HpyF30I.Experimental data indicated that HpyF30I restriction endonuclease didnot cleave TCGA target with m5C and hm5C modification in both strands(FIGS. 4 and 6).

Methods and Materials

Digestion of dam⁺ or dam⁻ DNA by HpyF30I restriction endonuclease. Oneμg pSEAd8 DNA isolated from E. coli dam⁺ or dam⁻ strain and linearizedby Eam11051 REaze was digested in parallel with 10 units of HpyF30IREase in 30 μl reaction buffer Tango™ (33 mM Tris-acetate; 10 mM Mgacetate; 66 mM K-acetate; 0.1 mg/ml BSA; pH 7.9 at 37° C.) at 37° C. forone h and with TaqI REase in reaction buffer TaqI (10 mM Tris-HCl; 5 mMMgCl₂; 100 mM NaCl; 0.1 mg/ml BSA; pH 8.0 at 37° C.) at 65° C. for oneh. The reactions were stopped by adding 6×DNA Loading Dye & SDS Solutionand resolved on 2% agarose gel in TBE buffer. 1351 bp DNA fragmentobtained after digestion with both REases of dam⁺ DNA (marked byasterisk) contains one TCGA sequence overlapping with dam sequence GATCin one strand and one TCGA sequence overlapping with dam sequence inboth strands. Both HpyF30I and TaqI cleaved these sites in dam⁻ DNAgenerating 289, 502 and 561 bp fragments (marked by asterisks).

Digestion of double stranded DNA oligonucleotides without and with m5Cmodification within TCGA site by HpyF30I restriction endonuclease. 55 ntlength single stranded oligonucleotide (see FIG. 2B upper strand) wasannealed with 17 nt length oligonucleotide M13/pUC Forward (experiment(a)). In experiment (b) the 55 nt single stranded oligonucleotide hadm5C modification within the TCGA sequence. The 5′-overhang was filled inby T4 DNA polymerase. Reactions were performed in 67 μl buffer Tango™containing 0.33 nmol of annealed oligonucleotides, dATP, dTTP and dGTP(0.5 mM of each), 17 u of T4 DNA polymerase and (a) 0.5 mM dCTP or (b)0.5 mM dm5CTP at 11° C. for 30 min and stopped by heating at 75° C. for10 min. Equal amounts of obtained double-stranded oligonucleotides (1 μgin each reaction) were digested with increasing amounts of HpyF30I in 20μl of buffer Tango™ at 37° C. for one h. Reactions were stopped byadding 6×DNA Loading Dye & SDS Solution and 10 μl of each were analyzedon 10% polyacrylamide gel in TBE buffer.

Digestion of in vitro methylated (M.SssI methyltransferase) lambda DNA(dam⁻) by HpyF30I restriction endonuclease. Lambda DNA (dam⁻) wasmodified in vitro by M.SssI DNA methyltransferase according tomanufacturer's recommendations(www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_em0821.pdf).After modification reaction DNA was extracted with chloroform andprecipitated with isopropanol (in the presence of 0.3 M NaCl). One μg ofobtained m5CpG modified lambda DNA as well as 1 μg of not modifiedlambda DNA (dam⁻) were digested in parallel with 10 u of HpyF30I REasein 20 μl reaction buffer SdaI (37 mM Tris-acetate; 15 mM Mg acetate; 150mM K-acetate; 0.1 mg/ml BSA; pH 7.0 at 37° C.) at 37° C. for one h andwith TaqI REase in reaction buffer TaqI at 65° C. for one h. Afterrestriction reaction mixtures were serially diluted in H₂O and qPCRreactions were performed on 5×10⁵ to 50 copies of not digested (controlreaction) or digested DNA in 25 μl reaction volume of Maxima SYBR GreenqPCR master Mix on Corbett Rotor-Gene 6000™ (Qiagen) instrument. 130 bpDNA fragment containing one TCGA site was amplified using primersLambda_fw (CTGATTCGTGGAACAGATACTC (SEQ ID NO: 27)) and Lambda_rw(ACACTTCAGGAGTGGAACGCA (SEQ ID NO: 28)).

Digestion of PCR fragments synthesized using dCTP, dm5CTP or dhm5CTP byHpyF30I restriction endonuclease. Three 962 bp double-stranded DNAfragments with 6 TCGA sequences were synthesized from plasmid DNAtemplate in PCR reaction mixtures containing buffer for Pfu DNApolymerase, recommended amount of Pfu DNA Polymerase, dATP, dTTP anddGTP, 0.2 mM each and (a) 0.2 mM dCTP, (b) 0.2 mM dm5CTP or (c) 0.2 mMdhm5CTP. PCR products were purified using GeneJET™ PCR Purification Kit.One μg of each PCR fragments were digested in parallel with 5 units ofHpyF30I REase in 30 μl of reaction buffer Tango™ at 37° C. for one h andwith 5 units of TaqI REase in reaction buffer TaqI at 65° C. for one h.The reactions were stopped by adding 6×DNA Loading Dye & SDS Solutionand resolved on 3% agarose gel in TBE buffer.

Example 3 The Interrogation of DNA Methylation Level within TCGA Targetin Human Jurkat Cell Line Genomic DNA Using HpyF30I and TaqI RestrictionEndonucleases

Spiked PCR Fragment Digestion Analysis in the Presence of Human JurkatCell Line Genomic DNA

Suitability of HpyF30I and TaqI restriction endonucleases formethylation analysis of genomic DNA was first evaluated using eithermethylated or unmethylated DNA as a spike control together with constantamount (0.5 μg) of human Jurkat cell line genomic DNA. The PCR fragmentwhich was amplified from lambda DNA in presence of either 100% of dCTPor dm5CTP in reaction mixtures subsequently was used as qPCR amplicon toevaluate digestion efficiency by HpyF30I and TaqI restrictionendonucleases in the presence of genomic DNA (FIGS. 7A to 7C).Methylated DNA was completely resistant to HpyF30I cleavage (100% ofresidual DNA target amplification after restriction reaction) meanwhileonly minor amounts of undigested target were detected (0.7%) aftertreatment with TaqI restriction endonuclease. In the same experimentunmodified DNA target was efficiently digested with both HpyF30I (7% ofresidual DNA target left) and TaqI (0.7%) enzymes. The data indicatedthat analysis of methylated DNA could be successfully performed in thepresence of genomic DNA maintaining the reaction conditions close to theexperiment where only genomic DNA would be digested and analyzed.

Methylation Analysis of DAPK1 Gene in Human Jurkat Cell Line Genomic DNAUsing HpyF30I/TaqI/PaeR7I Restriction and Subsequent qPCR

The methylation analysis of DAPK1 gene promoter region in human Jurkatcell line genomic DNA was performed using the same basic scheme as inall previous experiments. CTCGAG target present in DAPK1 gene promoterregion was addressed for restriction analysis with HpyF30I and TaqIenzymes (recognize inner tetranucleotide TCGA) and PaeR7I enzyme(recognize CTCGAG). PaeR7I enzyme is sensitive to methylation of innercytosine of CTCGAG sequence (REBASE). In this case HpyF30I, TaqI, andPaeR7I cleaving sites overlap and digestion completeness could becompared in cases of PaeR7I and HpyF30I enzymes. The same digestionlevel for both enzymes was expected if HpyF30I was also sensitive toinner cytosine methylation within CTCGAG target present in DAPK1 genepromoter region. All the restriction endonucleases HpyF30I, PaeR7I andTaqI almost completely cleaved their target leaving 2%, 0.8% and 0.7% ofundigested DNA, respectively (FIGS. 8A and 8B). The data indicated thatinner cytosine in CTCGAG target present in DAPK1 gene promoter regionwas completely unmethylated.

Methylation Analysis of RASSFA1 Gene in Human Jurkat Cell Line GenomicDNA Using HpyF30I/TaqI/PaeR7I Restriction and Subsequent qPCR

The methylation analysis of RASSFA1 gene promoter region in human Jurkatcell line genomic DNA was performed using the same scheme as in previousexperiments with DAPK1 gene analysis. CTCGAG target present in RASSFA1gene promoter region was addressed for restriction analysis with HpyF30Iand TaqI enzymes (recognize inner tetranucleotide TCGA) and PaeR7Ienzyme (recognize CTCGAG). PaeR7I enzyme is sensitive to methylation ofinner cytosine of CTCGAG sequence (REBASE). In this case HpyF30I, TaqI,and PaeR7I cleaving sites overlap and digestion completeness wascompared for PaeR7I and HpyF30I enzymes. The same digestion level forboth enzymes was expected if HpaF30I was also sensitive to innercytosine methylation within CTCGAG target present in RASSFA1 gene. Bothrestriction endonucleases HpyF30I and PaeR7I performed partial DNAcleavage leaving 50% of undigested target. At the same time TaqIrestriction endonuclease, which is not sensitive to cytosinemodifications, completely digested tested DNA sample leaving only about0.8% of undigested DNA (FIGS. 9A and 9B). The data indicated that thelevel of inner cytosine methylation in CTCGAG target present in RASSFA1gene promoter region is about 50% as confirmed by digestion with HpyF30Iand PaeR7I restriction endonucleases.

Methods and Materials

Digestion of PCR fragments (synthesized using dCTP or dm5CTP) in thepresence of genomic DNA. 130 bp DNA fragments containing one TCGAsequence were synthesized from lambda DNA template in PCR with Pfu DNApolymerase using primers Lambda_fw (CTGATTCGTGGAACAGATACTC (SEQ ID NO:27)) and Lambda_rw (ACACTTCAGGAGTGGAACGCA (SEQ ID NO: 28)). The reactionmixtures contained buffer for Pfu DNA polymerase, recommended amount ofthe enzyme, dATP, dTTP and dGTP, 0.2 mM each and 0.2 mM dCTP or 0.2 mMdm5CTP. PCR products were purified using GeneJET™ PCR Purification Kit.0.5 μg of methylated or unmethylated PCR fragment were mixed with 0.5 μgof human Jurkat cell line genomic DNA (purified with GeneJET™ GenomicDNA Purification Kit) and digested in parallel with 10 units of HpyF30IREase in 30 μl of reaction buffer SdaI at 37° C. for one h and with 10units of TaqI REase in reaction buffer TaqI at 65° C. for one h. Afterrestriction reaction mixtures were serially diluted in H₂O and qPCRreactions were performed on 5×10⁵ to 50 copies of not digested (controlreaction) or digested PCR fragment in 25 μl reaction volume of MaximaSYBR Green qPCR master Mix on StepOnePlus™ (Applied Biosystems)instrument. Primers Lambda_fw and Lambda_rw were used in qPCR.

Methylation analysis of DAPK1 gene in Human Jurkat cell line genomic DNAusing HpyF30I/TaqI/PaeR7I restriction endonucleases. One μg of HumanJurkat cell line genomic DNA (purified using GeneJET™ Genomic DNAPurification Kit) was digested in parallel with 10 units of HpyF30IREase in 20 μl reaction buffer SdaI at 37° C. for one h, with 20 unitsof PaeR7I REase in 20 μl of reaction buffer “4” at 37° C. for one h andwith 10 units of TaqI REase in 20 μl of reaction buffer TaqI at 65° C.for one h. The reaction mixtures were then serially diluted in H₂O andqPCR reactions were performed on 3×10⁵ to 30 copies of not digested(control reaction) or digested DNA in 25 μl reaction volume of MaximaSYBR Green qPCR master Mix on StepOnePlus™ (Applied Biosystems)instrument. 106 bp DNA fragment from DAPK1 gene promoter regioncontaining one CTCGAG sequence was amplified using primers DAPK_Tq_fw1(CTTTTGCTTTCCCAGCCAGG (SEQ ID NO: 29)) and DAPK_Tq_rw1(GATCGCACTTCTCCCCGAAG (SEQ ID NO: 30)).

Methylation analysis of RASSFA1 gene in Human Jurkat cell line genomicDNA using HpyF30I/TaqI/PaeR7I restriction endonucleases. In thisexperiment not digested or HpyF30I/TaqI/PaeR7I digested human Jurkatcell line genomic DNA was amplified using primers complementary to ofRASSFA1 gene promoter region: Rasf_fw2 (AAGATCACGGTCCAGCCTCT (SEQ ID NO:31)) and Rasf_rev2 (GCAACACACTTGGCCTACC (SEQ ID NO: 32)). 150 bp qPCRamplicon contains one CTCGAG sequence.

Example 4 The Interrogation of DNA Methylation Level within TCGA Targetin RASSFA1 Gene of Human Jurkat Cell Line Genomic DNA Using BisulphiteTreatment

To additionally validate that HpyF30I restriction endonuclease can beused for DNA methylation status determination within TCGA target, abisulfite treatment assay was used. During bisulfite treatment cytosinesare converted to uracyl if they are non methylated, while methylatedcytosines remain unchanged. Genomic DNA from human Jurkat cell line wastreated with sodium bisulfite. Target DNA fragment, namely the RASSFA1gene promoter region containing TCGA sequence, was then amplified usingTaq DNA polymerase and primers specific for converted DNA. The obtained164 bp length PCR product was cloned into pTZ57R/T cloning vector and 44individual clones were sequenced and analyzed. Sequence data showed that20 clones (about 45%) contained cytosines in TCGA sequence indicatingthat these targets are methylated and thus resistant to bisulfiteconversion. In the remaining 24 clones (about 55%) cytosines in TCGAsequence were converted to uracils and were read as thymines in thesequencing reads indicating that they were unmodified in the originalgenomic DNA.

The bisulfite sequencing experiment results correlated well with thedata obtained in the previous experiment, where human Jurkat cell linegenomic DNA was cleaved with HpyF30I restriction endonuclease anddigestion level of the same TCGA target in RASSFA1 gene promoter regionwas analyzed in subsequent qPCR. HpyF30I digestion resulted in about 50%of not cleaved target, thus indicating about 50% methylation levelwithin the target (FIGS. 9A and 9B). These observations confirmed thatHpyF30I digestion could be used to determine the methylation status ofTCGA sequences in eukaryotic genomic DNA.

Methods and Materials

The Conversion Reagent was prepared as follows: 0.9 ml sterile distilledwater was mixed with 200 μl 3M NaOH solution and 60 μl dimethylformamide(DMF). Sodium metabisulfite (0.72 g) was added to the solution anddissolved by inverting and shaking for about three min. PreparedConversion Reagent (120 μl) was added to 475 ng/5 μl DNA solution. Thereaction mixture was heated at 98° C. for ten min and subsequentlyincubated at 60° C. for three h. The bisulfite-treated DNA was purifiedusing EZ Bisulphite DNA Clean-up Kit™ (ZymoResearch).

Purified DNA (3 μl) was used as a template in the amplification reactionwith Taq DNA Polymerase (#EP0401) using primers konv_dir(AAATACACCCCTAAACCTCAAAATC (SEQ ID NO: 33)) and konv_rev(ATATTTGGTTTATTTATTGGGTGGG (SEQ ID NO: 34)) in 50 μl reaction mixturealso containing 0.2 mM dNTP Mix and PCR buffer (#838). The sample wasincubated at 95° C. for five min, and then 30 cycles of PCR at 94° C.for 30 s, 55° C. for 30 s and 72° C. for 30 s were performed. Theobtained 164 bp length PCR product was purified using GeneJET™ PCRPurification Kit and cloned into pTZ57R/T cloning vector usingInsTAclone™ PCR Cloning Kit. Plasmid DNAs were purified from 44individual colonies and cloned fragments Were sequenced using M13/pUCreverse sequencing primer (GAGCGGATAACAATTTCACACAGG (SEQ ID NO: 35)).

Example 5 HpyF30I Restriction Endonuclease is Sensitive to Glucosylatedhm5C

To demonstrate the sensitivity of the enzyme to glucosylated hm5C, PCRfragments containing two TCGA sequences were synthesized using dCTP ordhm5CTP. Half were treated with T4 β-glucosyltransferase (T4-BGT) whichadds a glucose moiety to 5-hydroxymethylcytosine. The DNA substrateswere then incubated with restriction enzymes. The PCR fragmentcontaining C was digested with HpyF30I restriction endonuclease whilethe fragment containing hm5C or glucosylated hm5C was completelyresistant to HpyF30I treatment (FIG. 10). All DNA samples were cleavedby TaqI restriction endonuclease which is not sensitive to C basemodifications within the TCGA sequence. The glucosylation reaction wasverified by digestion of DNA with FastDigest Mfel (the PCR fragment hasone target for this REase) which is not sensitive to hm5C modificationin CAATTG sequence but does not cleave DNA when hm5C is glucosylated(FIG. 10).

Methods and Materials

Digestion of PCR fragments (synthesized using dCTP or dhm5CTP) aftertreatment with T4 BGT. 1095 bp DNA fragments containing two TCGAsequences and one CAATTG sequence (FastDigest™Mfel target) weresynthesized from φX174 DNA template in PCR with DreamTaq DNA polymeraseusing primers X174_for (CACGCCAGAATACGAAAGACCAG (SEQ ID NO: 36)) andX174_rev (CGATAAACCAACCATCAGCATGAG (SEQ ID NO: 37)). The reactionmixtures contained buffer for DreamTaq DNA polymerase, recommendedamount of the enzyme, dATP, dTTP and dGTP, 0.2 mM each and 0.2 mM dCTPor 0.2 mM dhm5CTP. The PCR products were purified using GeneJET™ PCRPurification Kit. 2.5 μg of each PCR fragment were incubated in 150 μlof Epi Buffer with UDP-Glucose and T4 BGT according to manufacturer'srecommendations(http://www.fermentas.com/en/products/all/epigenetics/kl481-epijet-5-hmc-analysis-kit).Control reactions were incubated without T4 BGT. After heat inactivationof T4 BGT at 65° C. for ten min, the reaction mixtures were divided intofour equal parts and separate samples were incubated with 20 units ofTaqI REase at 65° C. for one h, 10 units of HpyF30I REase or 3 μl ofFastDigest™Mfel at 37° C. for one h. Control reactions were incubatedwithout enzymes. The restriction digestion products were then analyzedon 2% agarose gel in TBE buffer.

The following references are expressly incorporated herein by referencein their entirety:

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While the invention has been illustrated by a description of variousembodiments and while these embodiments have been described in detail,it is not Applicant's intention to restrict or in any way limit thescope of the appended claims to such detail. Additional advantages andmodifications will readily appear to those skilled in the art. Theinvention in its broader aspects is therefore not limited to thespecific details, representative enzymes, and methods, and illustrativeexamples shown and described. Accordingly, departures may be made fromsuch details without departing from the spirit or scope of the claims.

Applicants incorporate by reference the material contained in theaccompanying computer readable Sequence Listing identified asSequence_Listing_ST25.txt, having a file creation date of Jun. 25, 2013,2:06 p.m. and file size of 40.0 KB.

What is claimed is:
 1. A method for determining the level of methylationin eukaryotic double stranded DNA, the method comprising contacting afirst sample of the eukaryotic double stranded DNA with a restrictionendonuclease comprising SEQ ID NO. 4 or a sequence at least 70%identical to SEQ ID NO. 4, having a recognition sequence 5′-TCGA-3′where a modified cytosine residue in the recognition sequence impairscleavage, digesting the DNA comprising the recognition sequence5′-TCGA-3′ in which the cytosine residue is unmodified; and determiningthe amount of undigested DNA or digested DNA.
 2. The method of claim 1further comprising contacting a second sample of the double stranded DNAwith a second restriction endonuclease; and determining the amount ofDNA digestion, where the second restriction endonuclease has arecognition sequence 5′-TCGA-3′ and is not impaired by modification ofthe cytosine residue.
 3. The method of claim 1 wherein the eukaryoticdouble stranded DNA is genomic DNA.
 4. The method of claim 1 wherein theamount of undigested DNA is determined using qPCR.
 5. The method ofclaim 2 where the second restriction endonuclease is TaqI.
 6. The methodof claim 1 wherein the impaired ability to cleave the recognitionsequence means that under conditions at which the restrictionendonuclease is most active, optimal reaction conditions, the presenceof the modified cytosine residue in the recognition sequence reduces theendonuclease's ability to cleave the sequence by more than 80%.
 7. Themethod of claim 6 wherein the presence of the modified cytosine residuein the recognition sequence reduces the endonuclease's ability to cleavethe sequence by more than 90%.
 8. The method of claim 6 wherein thepercent of DNA cleavage is determined by qPCR analysis.
 9. The method ofclaim 1 wherein the modified cytosine residue is a methylated cytosine.10. The method of claim 9 wherein the methylated cytosine is selectedfrom the group consisting of 5-methylcytosine, 5-hydroxymethylcytosine,and glycosylated 5-hydroxymethylcytosine.